ObjectiveTo optimize the flow cytometric sorting workflow for myeloid-derived suppressor cells （MDSCs） from murine Lewis lung carcinoma （LLC） subcutaneous tumor tissues， improving single-cell suspension preparation efficiency and sorting purity， thereby providing high-quality cells for subsequent functional studies. MethodsSubcutaneous LLC tumor tissues were harvested from C57BL/6 mice， and two enzymatic digestion protocols were first compared to evaluate their effects on single-cell suspension preparation. After staining with a viability dye （Live/Dead） and fluorochrome-conjugated anti-mouse antibodies against CD45， CD11b， and Gr-1， cells were loaded onto a Beckman CytoFLEX cell sorter. Next， conventional and optimized gating strategies were applied. In the optimized strategy， the CD45⁺ population was used as the initial gate to directly define an MDSC-enriched region， followed by selection of CD11b⁺Gr-1⁺ cells. Finally， purity was assessed by flow cytometry， viability was determined by trypan blue staining， and RT-qPCR was performed to measure the expression of MDSC signature genes， including Arg1， Nos2， IL-10， and S100A8/A9. ResultsThe optimized enzymatic digestion protocol significantly increased the yield and viability of single cells. After optimization of the gating strategy， the post-sort positivity rate （purity） was markedly improved. Arg1， Nos2， IL-10 and S100A8/A9 were highly expressed in the sorted cells， indicating that the isolated cells retained characteristic MDSC features and functional signatures. ConclusionBy optimizing both the tissue digestion protocol and the flow cytometric gating strategy， we establish an efficient workflow for isolating MDSCs from tumor tissues. This method improves the yield and purity of single-cell preparations while preserving antigen integrity， providing a reliable methodological foundation for subsequent studies on MDSC function and metabolism.

Objective To observe the value of spectral CT material separation technology for diagnosing traumatic bone marrow edema in limbs.Methods Totally 51 patients with limb traumatic bone marrow edema were retrospectively enrolled and divided into young group(n=26,18-43 years)and middle-aged group(n=25,46-74 years).Taken MRI as reference standard,the efficacy of spectral CT Water-hydroxyapatite(HAP)image for diagnosing bone marrow edema in trauma area was analyzed,and the Water-HAP density values were compared between groups.Results No significant difference of diagnosing bone marrow edema was found between spectral CT and MRI(x2=0.201,P=0.654),and the consistency was high(Kappa=0.774).Water-HAP density value in bone marrow edema area was higher than that in non bone marrow edema area(t=24.634,P＜0.05),and no significant difference of Water-HAP density values in bone marrow edema area nor non bone marrow edema area was found between young group and middle-aged group(both P＞0.05).Conclusion Spectral CT material separation technology was helpful for diagnosing traumatic bone marrow edema in limbs.

ObjectiveTo explore the effects of Bushen Tiaojing Formula （补肾调经方） on the quality of early embryos in repeated controlled ovarian stimulation （COS） and its possible mechanism. MethodsA total of 150 ICR female mice were divided into a normal group， a model group， an inhibitor group， a low-dose and a high-dose Bushen Tiaojing Formula group. Mice in the normal group， inhibitor group and model group were gavaged with distilled water 0.25 ml a day， and mice in the low- and high-dose groups were given 25.6 and 51.2 g/（kg·d） of Bushen Tiaojing Formula， respectively. All groups were gavaged once a day for 13 days as a cycle. Mice in the normal group were injected intraperitoneally with 0.1 ml normal saline on the 11th day of gavage， while mice in the other four groups were used to establish COS models. The inhibitor group was injected with the endoplasmic reticulum stress inhibitor 4-phenylbutyric acid 0.2 ml one hour before modelling. All groups were injected by gavage and intraperitoneal injection for 3 consecutive cycles， with an interval of 4 days between cycles. Immediately after the third intraperitoneal injection， mice were grouped with male mice of the same strain in a ratio of 2∶1， and at 8AM of the second day， the mice were examined， and those with spermatozoa or spermatozoa in the vaginal smears were recorded as pregnant. The mice were executed in the afternoon of the 4th day of pregnancy， and the blastocysts were obtained under an inverted microscope. The morphology of the blastocysts was observed， and the total number of blastocysts and the number of high-quality blastocysts were recorded to calculate the rate of high-quality blastocysts； the apoptosis of blastocyst cells was detected by the Tunel method， and the apoptosis rate was calculated； the endoplasmic reticulum molecular chaperone protein glucose-regulated protein 78 （GRP78）， C/EBP homologous protein （CHOP）， B-lymphoblastoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， cystathionin 3 （Caspase-3） and cystathionin 12 （Caspase-12） protein levels and their mRNA expression were detected by immunofluorescent assay and RT-qPCR assay， respectively. ResultsIn the normal group， blastocysts had regular morphology， good morphological development and low fragmentation rate； in the model group， blastocysts were poorly developed， cell morphology was irregular， fragmentation rate was high， and there was some stagnation； in the inhibitor group and high-dose， low-dose Bushen Tiaojing Formula groups， cell morphology could be seen to be better developed， with regular morphology and less fragmentation rate. Compared with the normal group， the rate of high-quality blastocysts in the model group reduced， the rate of apoptosis of blastocyst cells increased， the levels of GRP78， CHOP， Caspase-12， Caspase-3， Bax protein and its mRNA expression in blastocysts increased， while the level of Bcl-2 protein and its mRNA reduced （P＜0.01）. Compared with the model group， the rate of high-quality blastocysts increased， the rate of apoptosis of blastocysts decreased， the levels of GRP78， CHOP， Caspase-12， Caspase-3， Bax protein and their mRNA expression decreased， and the levels of Bcl-2 protein and its mRNA expression increased in the blastocysts in the inhibitor group and the high-dose and low-dose Bushen Tiaojing Formula groups （P＜0.01）. Compared with the inhibitor group， GRP78， CHOP， Caspase-12， Bax protein levels and their mRNA expressions increased in the high-dose Bushen Tiaojing Formula group， and Caspase-12， Caspase-3， Bax protein levels and their mRNA expressions increased in the low-dose formula group （P＜0.05 or P＜0.01）. ConclusionBushen Tiaojing Formula can inhibit the apoptosis of blastocyst cells caused by repeated controlled ovarian stimulation by improving endoplasmic reticulum stress， which plays a role in improving the quality of early embryos.

Screening carbonyl reductases with the ability to catalyze the reduction of complex carbonyl compounds is of great significance for the biosynthesis of R-tolvaptan(R-TVP). In this study, the target carbonyl reductase in the crude enzyme extract of rabbit liver was separated, purified, and identified by ammonium sulfate precipitation, gel-filtration chromatography, ion exchange chromatography, affinity chromatography, and protein mass spectrometry. With the rabbit liver genome as the template, the gene encoding the carbonyl reductase rlsr5 was amplified by PCR and the recombinant strain was successfully constructed. After RLSR5 was purified by affinity chromatography, its enzymatic properties were characterized. The results indicated that the gene sequence of rlsr5 was 972 bp, encoding a protein with a molecular weight of 40 kDa. RLSR5 was a dimeric protein, and each monomer was composed of a (α/β)₈-barrel structure. RLSR5 could asymmetrically reduce 7-chloro-1-[2-methyl-4-[(2- methylbenzoyl)amino]benzoyl]-5-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine (prochiral ketone, PK) to synthesize R-TVP. The specific activity of the enzyme was 36.64 U/mg, and the optical purity of the product was 99%. This enzyme showcased the optimal performance at pH 6.0 and 30 °C. It was independent of metal ions, with the activity enhanced by Mn²⁺. This study lays a foundation for the biosynthesis of tolvaptan of optical grade.
Animals ; Rabbits ; Alcohol Oxidoreductases/biosynthesis* ; Recombinant Proteins/metabolism* ; Escherichia coli/metabolism* ; Liver/enzymology*

Objective To compare the efficacy of low-frequency and high-frequency repetitive transcranial magnetic stimulation(rTMS)in patients with tremor-dominant Parkinson's disease.Methods A total of 88 tremor-dominant Parkinson's disease patients who visited Suqian Hospital of Nanjing Drum Tower Hospital Group from January 2021 to June 2024 were randomly assigned to low-frequency rTMS group(treated by 1 Hz stimulation)and high-frequency rTMS group(treated by 5 Hz stimulation),with 44 patients in each group.Treatment was administered once a day,five times a week,for 4 weeks.Another 50 patients were enrolled as the control group.During the rTMS treatment,regular medications were continuously administrated.Before and after treatment,the following assessments were conducted:unified parkinson's disease rating scale part Ⅲ(UPDRS-Ⅲ),Parkinson's disease questionnaire-39(PDQ-39),timed up and go(TUG)test,tremor amplitude,tremor peak frequency,and cortical excitability(resting motion threshold[rMT]and active motion threshold[aMT]).Results After treatment,there were significant differences in the UPDRS-Ⅲ score,TUG time,PDQ-39 score,rMT and aMT values,and tremor amplitude between the two groups(P<0.05),and the high-frequency rTMS group was lower/shorter than the other two group.There was no significant difference in tremor peak frequency between the two groups(P>0.05).Conclusion Both low-frequency and high-frequency rTMS can significantly improve motor function and quality of life in tremor-dominant Parkinson's disease patients.High-frequency rTMS demonstrates better efficacy in improving various symptoms and provides a more effective treatment option for tremor-dominant Parkinson's disease.

ObjectiveTo investigate the effect and potential mechanism of caffeic acid （CA） on severe acute pancreatitis （SAP） induced by caerulein combined with lipopolysaccharide （LPS）， and to provide a basis for the research on novel drugs for the treatment of SAP. MethodsC57BL/6J mice， aged 6 weeks， were divided into control group， model group， CA group， and octreotide acetate （OA） group， with 6 mice in each group. The mice in the control group were given injection of normal saline， and those in the other groups were given intraperitoneal injection of caerulein combined with LPS to establish a mouse model of SAP. At 1 hour after the first injection of caerulein， the mice in the CA group and the OA group were given intraperitoneal injection of CA or subcutaneous injection of OA at an interval of 8 hours. The general status of the mice was observed after 24 hours of modeling， and serum， pancreas， lung， and colon samples were collected. HE staining was used to observe the histopathological changes of the pancreas and lungs， and the serum levels of α-amylase， lipase， tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， alanine aminotransferase， aspartate aminotransferase， and creatinine were measured. RT-PCR was used to measure the expression of proinflammatory factors in the pancreas and lungs； myeloperoxidase （MPO） immunohistochemistry was used to observe the degree of neutrophil infiltration； Western blot was used to measure the activation of nuclear factor-kappa B （NF-κB） and the level of citrullinated histone H3 （CitH3）， a marker for the formation of neutrophil extracellular traps （NETs）， in the pancreas and lungs， as well as the expression level of ZO-1 in colon tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the Dunnett’s t-test was used for further comparison between two groups. ResultsCompared with the control group， the model group had severe injury in the pancreas and lungs and significant increases in the activity of serum α- amylase and lipase and the levels of the proinflammatory cytokines IL-6， interleukin-1β （IL-1β）， and TNF-α in serum and lung tissue （all P<0.05）， as well as significant increases in NF-κB activation， neutrophil infiltration， and the formation of NETs in the pancreas and lungs （all P<0.05）. Compared with the model group， the CA group had alleviated pathological injury of the pancreas and lungs and significant reductions in the activity of serum α-amylase and the levels of the proinflammatory cytokines IL-6， IL-1β， and TNF-α in serum and lung tissue （all P<0.05）， as well as significant reductions in NF-κB activation， neutrophil infiltration， and the formation of NETs in the pancreas and lungs （all P<0.05）. ConclusionCA can alleviate SAP induced by caerulein combined with LPS in mice， possibly by inhibiting neutrophil recruitment and the formation of NETs.

BACKGROUND:Mammary stem cells are vital for the development and homeostasis of mammary gland tissue.The occurrence of breast cancer has a close relationship with the mammary stem cells.Recent studies have shown that Hopx,as an important transcriptional regulator of morphogenesis and cell differentiation,has been confirmed to be expressed in a variety of adult stem cells such as nerves,intestines,hair follicles and lungs.However,its role in mammary stem cells has not been reported so far.OBJECTIVE:To investigate whether Hopx expression marks mammary stem cells.METHODS:(1) Female Hopx-LacZ transgenic mice aged 8 weeks were selected to detect the background expression of Hopx in breast tissue by β-galactosidase staining.(2) Female wild-type mice at 4,6,and 8 weeks of age and 14.5 days of gestation were selected for whole-tissue magenta staining and K14 and K8 immunofluorescence staining,respectively.(3) Female Hopx-CreERT2;Rosa26LacZ transgenic mice aged 8 weeks and 17.5 days of gestation were selected and stained with breast β-galactosidase.(4) The 4-week-old female Hopx-CreERT2;Rosa26LacZ transgenic mice were selected.The Cre/loxp system was activated by intraperitoneal injection of tamoxifen (once every other day,three times),and breast β-galactosidase staining was performed 4 weeks after injection.The 8-week-old female Hopx-CreERT2;Rosa26LacZ transgenic mice were selected.The Cre/loxp system was activated by intraperitoneal injection of tamoxifen (once every other day,three times),and breast β-galactosidase staining was performed 4 and 10 weeks after the last injection.(5) Female Hopx-CreERT2;Rosa26LacZ transgenic mice aged 8 weeks were selected.The Cre/loxp system was activated by intraperitoneal injection of tamoxifen (once every other day,three times).Hopx-CreERT2;Rosa26LacZ transgenic mice were pregnant 2 weeks after injection.The mammary tissue of mice at 17.5 days of the first pregnancy and 17.5 days of the third pregnancy was stained with β-galactosidase.RESULTS AND CONCLUSION:(1) The results of β-galactosidase staining showed that the mammary ducts of Hopx-LacZ transgenic mice at 8 weeks of age did contain Hopx-positive cells and were located in the basal epithelia,with a small number.(2) Whole-mount staining of mammary glands and immunofluorescence staining results exhibited that the mammary glands of mice had different characteristics with corresponding developmental stages such as puberty,maturity,and pregnancy,and underwent a series of complex epithelial remodeling processes.(3) The results of β-galactosylase staining showed that Hopx-labeled positive cells in the mammary duct of Hopx-CreERT2;Rosa26LacZ transgenic mice at 17.5 days of gestation increased compared with female Hopx-CreERT2;Rosa26LacZ transgenic mice at 8 weeks of age.(4) The results of β-galactosylase staining showed that the Hopx-labeled positive cells in the mammary glands of 4-and 8-week-old female Hopx-CreERT2;Rosa26LacZ transgenic mice after tamoxifen injection were located in the basal epithelium with a small number.(5) The results of β-galactosidase staining showed that Hopx-labeled positive cells in the mammary glands of mice at 17.5 days of the first and third gestation were located in the basal epithelia around the alveoli,and the number of Hopx-labeled positive cells at 17.5 days of the third gestation was more.(6) In conclusion,Hopx reporter-marked basal epithelial cells belong to dormant mammary stem cells,which are responsible for the growth of the mammary glands during pregnancy and contribute to acinar formation.

Objective:To evaluate the value of noninvasive assessment of the 13C-methacetin breath test (13C-MBT) based on infrared isotope spectrometry for liver function reserve in patients with nonalcoholic fatty liver disease (NAFLD). Methods:A total of 120 NAFLD patients met the diagnostic criteria,who admitted to Department of Liver Diseases of the First Affiliated Hospital of Xinjiang Medical University from January 2023 to January 2024,were prospectively selected. Patients were divided into three groups based on liver stiffness measurement (LSM) of FibroTouch:mild fibrosis group (LSM<7.0 kPa,n=40),moderate fibrosis group (7.0≤LSM<9.5 kPa) and severe fibrosis group (LSM≥9.5 kPa,n=40). Meanwhile,40 healthy subjects were selected as a healthy control group. All subjects underwent 13C-MBT and conventional liver function tests. The differences of 13C-MBT parameters and liver function indicators among various groups were compared,and the correlations between 13C-MBT parameters and the degree of liver fibrosis,and between liver function indexes and the degree of liver fibrosis were analyzed. Receiver operating characteristic (ROC) curve was used to evaluate the judgment ability of 13C-MBT parameters to the degree of liver fibrosis. Results:The 13C-MBT parameters in NAFLD patients were lower than those in healthy control group,and the differences were statistically significant (F=27.413,28.635,29.851,P<0.05). With the aggravation of liver fibrosis,13C-MBT parameters in NAFLD patients gradually decreased. The 13C-MBT parameters of severe fibrosis group were significantly lower than those in the mild and moderate fibrosis groups,with statistically significant differences (t=12.331,13.020,14.232,22.033,21.032,29.332,P<0.05),respectively. The 13C-MBT parameters were positively correlated with liver function indicators,and were negatively correlated with LSM,and the absolute values of the correlation coefficients were>0.5,all of them showed statistically significant differences (r=0.375,-0.875,P<0.05). The 13C-MBT parameters had higher sensitivity and specificity in judging the degree of liver fibrosis. Taking MVmax40 as an example,when the limit value was 9.5 kPa,the sensitivity was 86.3％,and the specificity was 83.8％,and the accuracy was 85.0％,and the area under curve (AUC) was 0.913. Conclusion:13C-MBT based on infrared isotope spectrometry is a non-invasive,safe,rapid and accurate detection method,which can reflect the liver function reserve and liver fibrosis degree of NAFLD patients,and has important clinical value for the diagnosis and treatment of NAFLD.

Objective:To evaluate the value of noninvasive assessment of the 13C-methacetin breath test (13C-MBT) based on infrared isotope spectrometry for liver function reserve in patients with nonalcoholic fatty liver disease (NAFLD). Methods:A total of 120 NAFLD patients met the diagnostic criteria,who admitted to Department of Liver Diseases of the First Affiliated Hospital of Xinjiang Medical University from January 2023 to January 2024,were prospectively selected. Patients were divided into three groups based on liver stiffness measurement (LSM) of FibroTouch:mild fibrosis group (LSM<7.0 kPa,n=40),moderate fibrosis group (7.0≤LSM<9.5 kPa) and severe fibrosis group (LSM≥9.5 kPa,n=40). Meanwhile,40 healthy subjects were selected as a healthy control group. All subjects underwent 13C-MBT and conventional liver function tests. The differences of 13C-MBT parameters and liver function indicators among various groups were compared,and the correlations between 13C-MBT parameters and the degree of liver fibrosis,and between liver function indexes and the degree of liver fibrosis were analyzed. Receiver operating characteristic (ROC) curve was used to evaluate the judgment ability of 13C-MBT parameters to the degree of liver fibrosis. Results:The 13C-MBT parameters in NAFLD patients were lower than those in healthy control group,and the differences were statistically significant (F=27.413,28.635,29.851,P<0.05). With the aggravation of liver fibrosis,13C-MBT parameters in NAFLD patients gradually decreased. The 13C-MBT parameters of severe fibrosis group were significantly lower than those in the mild and moderate fibrosis groups,with statistically significant differences (t=12.331,13.020,14.232,22.033,21.032,29.332,P<0.05),respectively. The 13C-MBT parameters were positively correlated with liver function indicators,and were negatively correlated with LSM,and the absolute values of the correlation coefficients were>0.5,all of them showed statistically significant differences (r=0.375,-0.875,P<0.05). The 13C-MBT parameters had higher sensitivity and specificity in judging the degree of liver fibrosis. Taking MVmax40 as an example,when the limit value was 9.5 kPa,the sensitivity was 86.3％,and the specificity was 83.8％,and the accuracy was 85.0％,and the area under curve (AUC) was 0.913. Conclusion:13C-MBT based on infrared isotope spectrometry is a non-invasive,safe,rapid and accurate detection method,which can reflect the liver function reserve and liver fibrosis degree of NAFLD patients,and has important clinical value for the diagnosis and treatment of NAFLD.

Objective To observe the value of spectral CT material separation technology for diagnosing traumatic bone marrow edema in limbs.Methods Totally 51 patients with limb traumatic bone marrow edema were retrospectively enrolled and divided into young group(n=26,18-43 years)and middle-aged group(n=25,46-74 years).Taken MRI as reference standard,the efficacy of spectral CT Water-hydroxyapatite(HAP)image for diagnosing bone marrow edema in trauma area was analyzed,and the Water-HAP density values were compared between groups.Results No significant difference of diagnosing bone marrow edema was found between spectral CT and MRI(x2=0.201,P=0.654),and the consistency was high(Kappa=0.774).Water-HAP density value in bone marrow edema area was higher than that in non bone marrow edema area(t=24.634,P＜0.05),and no significant difference of Water-HAP density values in bone marrow edema area nor non bone marrow edema area was found between young group and middle-aged group(both P＞0.05).Conclusion Spectral CT material separation technology was helpful for diagnosing traumatic bone marrow edema in limbs.